In vivo role of aldehyde reductase.

BACKGROUND: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. METHODS: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. RESULTS: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. CONCLUSIONS: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. GENERAL SIGNIFICANCE: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.

Vitamin C is not essential for carnitine biosynthesis in vivo: verification in vitamin C-depleted senescence marker protein-30/gluconolactonase knockout mice.

Carnitine is an essential cofactor in the transport of long-chain fatty acids into the mitochondrial matrix and plays an important role in energy production via beta-oxidation. Vitamin C (VC) has long been considered a requirement for the activities of two enzymes in the carnitine biosynthetic pathway, i.e., 6-N-trimethyllysine dioxygenase and gamma-butyrobetaine dioxygenase. Our present study using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo, led to the conclusion that this notion is not true. After weaning at 40 d of age, SMP30/GNL KO mice were fed a diet lacking VC and carnitine, then given water containing 1.5 g/l VC (VC(+) mice) or no VC (VC(-) mice) for 75 d. Subsequently, total VC and carnitine levels were measured in the cerebrum, cerebellum, liver, kidney, soleus muscle, extensor digitorum longus muscle, heart, plasma and serum. The total VC levels in all tissues and plasma from VC(-) SMP30/GNL KO mice were negligible, i.e., <2% of the levels in SMP30/GNL KO VC(+) mice; however, the total carnitine levels of both groups were similar in all tissues and serum. In addition, carnitine was produced by incubated liver homogenates from the VC-depleted SMP30/GNL KO mice irrespective of the presence or absence of 1 mM VC. Collectively, these results indicate that VC is not essential for carnitine biosynthesis in vivo.

Combination of NADPH and copper ions generates proteinase K-resistant aggregates from recombinant prion protein.

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23-98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3'-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23-98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23-231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.

Senescence marker protein 30 functions as gluconolactonase in L-ascorbic acid biosynthesis, and its knockout mice are prone to scurvy.

We originally identified senescence marker protein 30 (SMP30) as a distinctive protein whose expression decreases in an androgen-independent manner with aging. Here, we report its sequence homology found in two kinds of bacterial gluconolactonases (GNLs) by using the blast search. Then, through a biochemical study, we identify SMP30 as the lactone-hydrolyzing enzyme GNL of animal species. SMP30 purified from the rat liver had lactonase activity toward various aldonolactones, such as d- and l-glucono-delta-lactone, d- and l-gulono-gamma-lactone, and d- and l-galactono-gamma-lactone, with a requirement for Zn(2+) or Mn(2+) as a cofactor. Furthermore, in SMP30 knockout mice, no GNL activity was detectable in the liver. Thus, we conclude that SMP30 is a unique GNL in the liver. The lactonase reaction with l-gulono-gamma-lactone is the penultimate step in l-ascorbic acid (AA) biosynthesis, and the essential role of SMP30 in this synthetic process was verified here by a nutritional study using SMP30 knockout mice. These knockout mice (n = 6), fed a vitamin C-deficient diet, did not thrive; i.e., they displayed symptoms of scurvy such as bone fracture and rachitic rosary and then died by 135 days after the start of receiving the deficient diet. The AA levels in their livers and kidneys at the time of death were <1.6% of those in WT control mice. In addition, by using the SMP30 knockout mouse, we demonstrate that the alternative pathway of AA synthesis involving d-glucurono-gamma-lactone operates in vivo, although its flux is fairly small.

Enhanced oxidative stress by L-ascorbic acid within cells challenged by hydrogen peroxide.

It has been amply documented that L-ascorbic acid added to the medium of a cell culture increases oxidative damage, and this effect of L-ascorbic acid has been ascribed to the generation of reactive oxygen intermediates in the medium during its auto-oxidation. We have here questioned whether such an effect is exerted inside the cell as well, and if so, what its mechanism is. To assess thiol oxidation in the cell, we manipulated CHO cells so that they could express bacterial alkaline phosphatase in the cytoplasm. Alkaline phosphatase activity, which requires the formation of intramolecular disulfide bridges, was shown to appear when the cells were exposed to H2O2. This H2O2-induced activity increased more than 1.5 fold when L-ascorbic acid had been loaded in the cells by incubation with L-ascorbic acid-2-O-phosphate. Similar enhancing effects were also observed by assessing oxidation of glutathione, formation of protein carbonyls, and generation of reactive oxygen intermediates. Interestingly, the effects by the L-ascorbic acid-2-O-phosphate treatment were totally suppressed by addition of the membrane-permeable chelator deferoxamine to the medium, indicating the involvement of iron ions. Because the apoprotein of conalbumin, which binds iron ions with a high affinity, had no effect and because the same deferoxamine effect was observed with the cells incubated in balanced salt solution with no metal salts added, it was concluded that L-ascorbic acid acts as a pro-oxidant within the cell suffering oxidative stress, and that this effect is elicited through increased redox-cycling of iron in combination with L-ascorbic acid.

Fragmentation and dimerization of copper-loaded prion protein by copper-catalysed oxidation.

Prion protein consists of an N-terminal domain containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ and a C-terminal domain composed of three alpha-helices and two short beta-strands. Several studies have shown that the N-terminal domain binds five Cu2+ ions. In the present study, we have investigated copper-catalysed oxidation of a recombinant mouse prion protein, PrP23-231. The copper-loaded PrP23-231 was found to be carbonylated by incubation with dopamine. Besides the formation of carbonyls, a cross-linked species with the dimeric size and C-terminally truncated species were generated. These reactions were retarded in the presence of Cu+- and Cu2+-specific copper chelators, catalase, and SOD (superoxide dismutase), but not in the presence of various bivalent metal ions. Together, these results indicate that the copper bound to prion protein undergoes catalytic cycling in the presence of catecholamines and causes the oxidation of the protein.

The whole structure of the human nonfunctional L-gulono-gamma-lactone oxidase gene--the gene responsible for scurvy--and the evolution of repetitive sequences thereon.

L-Gulono-gamma-lactone oxidase (GULO), which catalyzes the last step of ascorbic acid biosynthesis, is missing in humans. The whole structure of the human gene homologue for this enzyme was disclosed by a computer-assisted search. Only five exons, as compared to 12 exons constituting the functional rat GULO gene, remain in the human genome. A comparison of these exons with those of their functional counterparts in rat showed that there are two single nucleotide deletions, one triple nucleotide deletion, and one single nucleotide insertion in the human sequence. When compared in terms of codons, the human sequence has a deletion of a single amino acid, two stop codons, and two aberrant codons missing one nucleotide besides many amino acid substitutions. A comparison of the remaining human exon sequences with the corresponding sequences of the guinea pig nonfunctional GULO gene revealed that the same substitutions from rats to both species occurred at a large number of nucleotide positions. From analyses of the molecular evolution of Alu sequences in the human GULO gene homologue, it is thought that two Alu sequences were inserted in the vicinity of a presumed position of lost exon 11 during the same period as GULO lost its function. It is predicted that six LINE-1 sequences located in and near the gene homologue were inserted not during that period.

Impaired ascorbic acid metabolism in streptozotocin-induced diabetic rats.

Ascorbic acid (AA) metabolism in streptozotocin (STZ)-induced diabetic rats was determined by examining urinary excretion, renal reabsorption, reductive regeneration, and biosynthesis of AA at 3 and 14 days after STZ administration. AA concentrations in the plasma, liver, and kidney of the diabetic rats were significantly lower than those of controls on d 3, and decreased further as the diabetic state continued. Hepatic AA regeneration significantly decreased in the diabetic rats on d 3 in spite of increased gene expressions of AA regenerating enzymes and was further reduced on d 14. Hepatic activity of L-gulono-gamma-lactone oxidase, a terminal enzyme of hepatic AA biosynthesis, also decreased significantly on d 3 and decreased further on d 14. Urinary excretion of AA was significantly increased on d 3, with an increase in urine volume but no change in gene expressions of renal AA transporters (SVCT1 and SVCT2). Urinary excretion of AA was normalized on d 14. The results suggest that impaired hepatic and renal regeneration, as well as increased urinary excretion and impaired hepatic biosynthesis of AA, contributed to the decrease in AA in plasma and tissues of STZ-induced diabetic rats.

Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly.

An Rpn9-disrupted yeast strain, Delta rpn9, whose growth is temperature sensitive with defective assembly of the 26 S proteasome complex, was studied. This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type. Nondenaturing gel electrophoresis followed by activity staining revealed that Delta rpn9 yeast cells had a higher activity of 20 S proteasome than wild type and that in both Delta rpn9 and wild-type cells treated with hydrogen peroxide, 20 S proteasome activity was increased with a concomitant decrease in 26 S proteasome activity. Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells. Taken together, these results suggest that the 20 S proteasome degrades oxidized proteins without ubiquitination of target proteins.

Carbonyl formation on a copper-bound prion protein fragment, PrP23-98, associated with its dopamine oxidase activity.

The amino-terminal part of prion protein (PrP), containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ, has been implicated in the binding of copper ion. This region possesses amino acid residues susceptible to oxidation, such as histidine, lysine, arginine and proline. In this study, we have investigated copper-catalyzed oxidation of an N-terminal part of human PrP, PrP23-98, that was prepared by the recombinant DNA technique. Carbonyl formations on copper-bound PrP23-98 induced by dopamine and L-ascorbate were analyzed kinetically, and it was found that the redox cycling of PrP23-98-bound copper, especially induced by dopamine, was coupled to the formation of carbonyls on the protein.